Isopycnic centrifugation

Isopycnic centrifugation or equilibrium centrifugation is a process used to isolate nucleic acids such as DNA. To begin the analysis a mixture of caesium chloride and DNA is placed in a centrifuge for several hours at high speed to generate a force of about 10^5 x g (earth's gravity). Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mol it is similar to the density of DNA. After some time a gradient of the caesium ions is formed, caused by two opposing forces: diffusion and centrifugal force. The diffusive force arises due to the density gradient of solvated caesium chloride and it always directed towards the center of the rotor. The sedimenting particles will sediment away from the rotor until their density is equivalent to the local density of the caesium gradient, at which point the diffusive force is equivalent to the centrifugal force.

The DNA molecules will then be separated based on the relative proportions of AT (adenine and thymine base pairs) to GC (guanine and cytosine base pairs). An AT base pair has a lower molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the one with the greater proportion of AT base pairs will have a lower density, all other factors being equal. Different types of nucleic acids will also be separated into bands, e.g. RNA is denser than supercoiled plasmid DNA, which is denser than linear chromosomal DNA.

References

• Gerald Karp, Cell and molecular biology: Concepts and experiments, fourth edition, 2005, Von Hoffman press

Isopycnic gradient ultracentrifugation is a process used frequently to separate plasma LPs (lipoproteins). Since lipids have lower buoyant densities than proteins, LPs containing higher % of lipid component relative to protein component will sediment on upper layers.

References

• Roger A. Davis and Jean E. Vance, Structure, assembly and secretion of lipoproteins, 1996, Elsevier Science B. V.